The Role Of Dmrt1 And Gsdf In Sex Determination And Differentiation In Spotted Scat,Scatophagus Argus

The sex control of fish has currently received all-out attention in the aquaculture industry.The spotted scat,Scatophagus argus(Linnaeus,1766)is a euryhaline fish which is being used as ornamental and food fish and has a 1:1 sex ratio in the cultured environment.Meanwhile,females grow significantly faster and larger than males,nonetheless,information on its sex control is limited.Hence cloning the master sex-determination(SD)gene will be helpful for its sex control in the aquaculture industry.This study sought to provide useful information that will contribute to the understanding of spotted scat sex determination and differentiation and the possibility for mono-sex(all males/female)fingerling production.Below summarizes the main findings of the study: 1.Identification of sex-specific markers and cloning of candidate sexdetermination gene in S.argusIn this study,two duplicates of Dmrt1(Dmrt1 and Dmrt1b)and Dmrt3(Dmrt3 and Dmrt3△-Y)were isolated and cloned from S.argus.Innovatively,two sex-linked markers,Dmrt3-Marker-F/R and Dmrt1-Marker-F/R were developed from DM-domain genes transcripts in S.argus.The single-nucleotide polymorphisms(SNP)at position-151 and 166 bp on the DNA amplified by Dmrt3-Marker-F/R are all homozygous CC GG genotype in females and heterozygous CG GA genotype in the males.Dmrt3-Marker-F/R was developed from the truncated Dmrt3 sequence to specifically amplify a portion of the genomic region which might cause the premature of the c DNA.These results indicate that the sexes of S.argus are controlled by sex chromosomes(XX females,XY males).The nucleotide at position 166 bp of the DNA amplified by Dmrt3-Marker-F/R is found to be a donor splice site mutation(c.400+1G > A)in Dmrt3 which generates a truncated isoform.The truncated Dmrt3 is linked to male sex and thereafter named as Dmrt3△-Y,and its allele is the normal Dmrt3 on the X chromosome.Also,a normal Dmrt1 and a truncated Dmrt1(Dmrt1b)were cloned.The Dmrt1 and Dmrt1 b encode proteins of 300 and 89 amino acid residues(aa),respectively.Multiple alignments of Dmrt1 s in vertebrates revealed that S.argus Dmrt1 and Dmrt1 b share a highly conserved DM domain region with other species.A pair of primer named Dmrt1-MarkerF/R whose forward and reverse primers are located on the putative exon 2 and exon 3 was able to amplify a fragment of 3.2 kb genomic DNA from male individuals.Sequence analysis revealed that Dmrt1-Marker-F/R located on Dmrt1 is specifically located on Chromosome Y,indicating that,the normal Dmrt1 is Y specific.A pair of primers named Dmrt1b-MarkerF/R developed on Dmrt1 b was used to amplify a fragment around 1.5 kb from the genomic DNA of both male and female with a similarity of more than 99.0 %.Dmrt1b-Marker-F/R located Dmrt1 b is not sex-specific and should be the X chromosome allele of Y specific Dmrt1.Dmrt3-Marker-F/R and Dmrt1-Marker-F/R were validated in more than Two hundred and seventy(270)and Two thousand(2000)individual male and female S.argus respectively from different populations in Guangdong province,China.2.Cloning and characterization of gonadal soma-derived factor(Gsdf)and its relationship with Dmrt1 in S.argusThe partial c DNA of S.argus Gsdf consisted of 820 nucleotides with a 654 bp ORF encoding 217 amino acid(aa)residues.S.argus Gsdf possessed conserved cysteine residues in its TGF-b domain.Tissue distribution analysis indicated that the S.argus Gsdf only expressed in gonadal tissues of which,it was expressed significantly higher in the testis than in the ovary.The expression levels of Gsdf,Dmrt1,and Sf1 in gonads at different developmental stages were analyzed by real-time PCR.Gsdf was expressed significantly lower in phase V testes than in the testes at phase III and IV,while it was expressed at much higher levels in phase V testes than in ovaries at phase II,III and IV.In ovaries,Gsdf was expressed highest at phase II.Dmrt1 was expressed significantly higher in testes(at phase III,IV and V)than in ovaries at phases II,III and IV.In females,Sf1 was expressed highest in ovaries at phase III,while sf1 was expressed similarly in testes at phase III,IV and V.Western blot analysis indicates that,S.argus Gsdf expressed extremely higher in testis than in the ovary.The testicular Gsdf specific band was undetectable when the pre-adsorption primary antibody was used.Immunohistochemistry(IHC)studies revealed that,Gsdf was exclusively expressed in the Sertoli cells surrounding the spermatogonia of the testes.The Gsdf signal could not be detected in the ovaries at higher dilution factor,while a relatively weaker signal was detected in ovaries at lower dilutions.In female fish,Gsdf was expressed exclusively in the somatic cells surrounding the oogonia of the ovary.In silico analyses of the S.argus Gsdf promoter revealed six Sf1 binding sites.Also,a luciferase assay revealed that Sf1 alone can activate Gsdf transcription,in a dose-dependent manner whiles Dmrt1 alone could not activate Gsdf gene transcription.When co-transfected with Sf1,Dmrt1 was able to activate Gsdf expression in a dose-dependent manner.These results strongly support that the putative binding sites for Dmrt1 or Sf1 are required for the regulation of spotted scat Gsdf gene transactivation.ConclusionTaking together,two sex-specific markers were developed in this study which revealed that XX-XY systems determine sex in S.argus.The Markers are located within Dmrt3 and Dmrt1 loci,respectively.The truncated Dmrt1 b and normal Dmrt3 are located on the X chromosome,while the normal Dmrt1 and truncated Dmrt3Δ-Y are located on the Y chromosome.The XX S.argus lack the functional Dmrt1,therefore its gonad will develop into an ovary.Between the Y specific Dmrt1 and Dmrt3Δ-Y,Dmrt1 should most probably be the candidate SD gene in XY S.argus.In addition,we cloned S.argus gsdf which shares common features with typical TGF-β superfamily members.Gsdf was predominantly expressed in the testes than ovaries in the S.argus.In vitro promoter analyses demonstrated that Dmrt1 promoted Gsdf expression in the presence of Sf1 in spotted scat.Our study also revealed a possible role for Gsdf in male sex differentiation under the regulation of Dmrt1 in the spotted scat.Our research provides insight into the sex control of S.argus in aquaculture.